bims-nucpor 2024-01-21 papers

bims-nucpor

Biomed News

on Nuclear pore complex and nucleoporins in stress, aging and disease

Issue of 2024–01–21
seven papers selected by
Sara Mingu, Johannes Gutenberg University

HIV-1 capsid shape, orientation, and entropic elasticity regulate translocation into the nuclear pore complex.
Nucleocytoplasmic transport rates are regulated by cellular processes that modulate GTP availability.
Quantitative comparison of nuclear transport inhibition by SARS coronavirus ORF6 reveals the importance of oligomerization.
Mechanism of exportin retention in the cell nucleus.
Sculpting nuclear envelope identity from the endoplasmic reticulum during the cell cycle.
HIV-1 Capsid Uncoating Is a Multistep Process That Proceeds through Defect Formation Followed by Disassembly of the Capsid Lattice.
Podocyte-specific Nup160 knockout mice develop nephrotic syndrome and glomerulosclerosis.

Proc Natl Acad Sci U S A. 2024 Jan 23. 121(4): e2313737121

HIV-1 capsid shape, orientation, and entropic elasticity regulate translocation into the nuclear pore complex.

Arpa Hudait, Gregory A Voth.

  Nuclear import and uncoating of the viral capsid are critical steps in the HIV-1 life cycle that serve to transport and release genomic material into the nucleus. Viral core import involves translocating the HIV-1 capsid at the nuclear pore complex (NPC). Notably, the central channel of the NPC appears to often accommodate and allow passage of intact HIV-1 capsid, though mechanistic details of the process remain to be fully understood. Here, we investigate the molecular interactions that operate in concert between the HIV-1 capsid and the NPC that regulate capsid translocation through the central channel. To this end, we develop a "bottom-up" coarse-grained (CG) model of the human NPC from recently released cryo-electron tomography structure and then construct composite membrane-embedded CG NPC models. We find that successful translocation from the cytoplasmic side to the NPC central channel is contingent on the compatibility of the capsid morphology and channel dimension and the proper orientation of the capsid approach to the channel from the cytoplasmic side. The translocation dynamics is driven by maximizing the contacts between phenylalanine-glycine nucleoporins at the central channel and the capsid. For the docked intact capsids, structural analysis reveals correlated striated patterns of lattice disorder likely related to the intrinsic capsid elasticity. Uncondensed genomic material inside the docked capsid augments the overall lattice disorder of the capsid. Our results suggest that the intrinsic "elasticity" can also aid the capsid to adapt to the stress and remain structurally intact during translocation.

Keywords:  HIV-1 capsid; antiviral drugs; coarse-grained molecular dynamics; nuclear pore complex; nucleocytoplasmic transport

DOI:  https://doi.org/10.1073/pnas.2313737121
bioRxiv. 2023 Dec 30. pii: 2023.12.29.573651. [Epub ahead of print]

Nucleocytoplasmic transport rates are regulated by cellular processes that modulate GTP availability.

Kelsey L Scott, Charles T Halfmann, Allison D Hoefakker, Purboja Purkayastha, Ting Ching Wang, Tanmay P Lele, Kyle J Roux.

Nucleocytoplasmic transport (NCT), the facilitated diffusion of cargo molecules between the nucleus and cytoplasm through nuclear pore complexes (NPCs), enables numerous fundamental eukaryotic cellular processes. Ran GTPase uses cellular energy in the direct form of GTP to create a gradient across the nuclear envelope (NE) that drives the majority of NCT. We report here that changes in GTP availability resulting from altered cellular physiology modulate the rate of NCT, as monitored using synthetic and natural cargo, and the dynamics of Ran itself. Cell migration, cell spreading and/or modulation of the cytoskeleton or its connection to the nucleus alter GTP availability and thus rates of NCT, regulating RNA export and protein synthesis. These findings support a model in which changes in cellular physiology that alter GTP availability can regulate the rate of NCT, impacting fundamental cellular processes that extensively utilize NCT.
Summary: Changes in the availability of cellular GTP resulting from physiologically relevant processes, including cell migration and cell spreading, alter the rates of Ran-dependent nuclear import and export. Altered rates of nucleocytoplasmic transport regulate RNA localization and protein synthesis.

DOI: https://doi.org/10.1101/2023.12.29.573651
Proc Natl Acad Sci U S A. 2024 Jan 23. 121(4): e2307997121

Quantitative comparison of nuclear transport inhibition by SARS coronavirus ORF6 reveals the importance of oligomerization.

Tae Yeon Yoo, Timothy J Mitchison.

  Open Reading Frame 6 (ORF6) proteins, which are unique to severe acute respiratory syndrome-related (SARS) coronavirus, inhibit the classical nuclear import pathway to antagonize host antiviral responses. Several alternative models were proposed to explain the inhibitory function of ORF6 [H. Xia et al., Cell Rep. 33, 108234 (2020); L. Miorin et al., Proc. Natl. Acad. Sci. U.S.A. 117, 28344-28354 (2020); and M. Frieman et al., J. Virol. 81, 9812-9824 (2007)]. To distinguish these models and build quantitative understanding of ORF6 function, we developed a method for scoring both ORF6 concentration and functional effect in single living cells. We combined quantification of untagged ORF6 expression level in single cells with optogenetics-based measurement of nuclear transport kinetics, using methods that could be adapted to measure concentration-dependent effects of any untagged protein. We found that SARS-CoV-2 ORF6 is ~15 times more potent than SARS-CoV-1 ORF6 in inhibiting nuclear import and export, due to differences in the C-terminal region that is required for the NUP98-RAE1 binding. The N-terminal region was required for transport inhibition. This region binds membranes but could be replaced by synthetic constructs which forced oligomerization in solution, suggesting its primary function is oligomerization. We propose that the hydrophobic N-terminal region drives oligomerization of ORF6 to multivalently cross-link the NUP98-RAE1 complexes at the nuclear pore complex, and this multivalent binding inhibits bidirectional transport.

Keywords:  ORF6; SARS-CoV-2; nuclear pore; nuclear transport

DOI:  https://doi.org/10.1073/pnas.2307997121
J Cell Biol. 2024 Feb 05. pii: e202306094. [Epub ahead of print]223(2):

Mechanism of exportin retention in the cell nucleus.

Larisa E Kapinos, Joanna Kalita, Elena Kassianidou, Chantal Rencurel, Roderick Y H Lim.

Exportin receptors are concentrated in the nucleus to transport essential cargoes out of it. A mislocalization of exportins to the cytoplasm is linked to disease. Hence, it is important to understand how their containment within the nucleus is regulated. Here, we have studied the nuclear efflux of exportin2 (cellular apoptosis susceptibility protein or CAS) that delivers karyopherinα (Kapα or importinα), the cargo adaptor for karyopherinβ1 (Kapβ1 or importinβ1), to the cytoplasm in a Ran guanosine triphosphate (RanGTP)-mediated manner. We show that the N-terminus of CAS attenuates the interaction of RanGTPase activating protein 1 (RanGAP1) with RanGTP to slow GTP hydrolysis, which suppresses CAS nuclear exit at nuclear pore complexes (NPCs). Strikingly, a single phosphomimetic mutation (T18D) at the CAS N-terminus is sufficient to abolish its nuclear retention and coincides with metastatic cellular behavior. Furthermore, downregulating Kapβ1 disrupts CAS nuclear retention, which highlights the balance between their respective functions that is essential for maintaining the Kapα transport cycle. Therefore, NPCs play a functional role in selectively partitioning exportins in the cell nucleus.

DOI: https://doi.org/10.1083/jcb.202306094
Nucleus. 2024 Dec;15(1): 2299632

Sculpting nuclear envelope identity from the endoplasmic reticulum during the cell cycle.

Pallavi Deolal, Julia Scholz, Kaike Ren, Helena Bragulat-Teixidor, Shotaro Otsuka.

  The nuclear envelope (NE) regulates nuclear functions, including transcription, nucleocytoplasmic transport, and protein quality control. While the outer membrane of the NE is directly continuous with the endoplasmic reticulum (ER), the NE has an overall distinct protein composition from the ER, which is crucial for its functions. During open mitosis in higher eukaryotes, the NE disassembles during mitotic entry and then reforms as a functional territory at the end of mitosis to reestablish nucleocytoplasmic compartmentalization. In this review, we examine the known mechanisms by which the functional NE reconstitutes from the mitotic ER in the continuous ER-NE endomembrane system during open mitosis. Furthermore, based on recent findings indicating that the NE possesses unique lipid metabolism and quality control mechanisms distinct from those of the ER, we explore the maintenance of NE identity and homeostasis during interphase. We also highlight the potential significance of membrane junctions between the ER and NE.

Keywords:  Cell cycle; ER–NE junction; endoplasmic reticulum; mitosis; nuclear assembly; nuclear envelope; nuclear pore complex

DOI:  https://doi.org/10.1080/19491034.2023.2299632
ACS Nano. 2024 Jan 19.

HIV-1 Capsid Uncoating Is a Multistep Process That Proceeds through Defect Formation Followed by Disassembly of the Capsid Lattice.

Levi B Gifford, Gregory B Melikyan.

  The HIV-1 core consists of a cone-shaped capsid shell made of capsid protein (CA) hexamers and pentamers encapsulating the viral genome. HIV-1 capsid disassembly, referred to as uncoating, is important for productive infection; however, the location, timing, and regulation of uncoating remain controversial. Here, we employ amber codon suppression to directly label CA. In addition, a fluid phase fluorescent probe is incorporated into the viral core to detect small defects in the capsid lattice. This double-labeling strategy enables the visualization of uncoating of single cores in vitro and in living cells, which we found to always proceed through at least two distinct steps─the formation of a defect in the capsid lattice that initiates gradual loss of CA below a detectable level. Importantly, intact cores containing the fluid phase and CA fluorescent markers enter and uncoat in the nucleus, as evidenced by a sequential loss of both markers, prior to establishing productive infection. This two-step uncoating process is observed in different cells, including a macrophage line. Notably, the lag between the release of fluid phase marker and terminal loss of CA appears to be independent of the cell type or reverse transcription and is much longer (>5-fold) for nuclear capsids compared to cell-free cores or cores in the cytosol, suggesting that the capsid lattice is stabilized by capsid-binding nuclear factors. Our results imply that intact HIV-1 cores enter the cell nucleus and that uncoating is initiated through a localized defect in the capsid lattice prior to a global loss of CA.

Keywords:  HIV-1 capsid; click labeling; dynamics of capsid uncoating; genetic code expansion; nuclear import; single virus tracking

DOI:  https://doi.org/10.1021/acsnano.3c07678
Hum Mol Genet. 2024 Jan 15. pii: ddad211. [Epub ahead of print]

Podocyte-specific Nup160 knockout mice develop nephrotic syndrome and glomerulosclerosis.

Yuanyuan Li, Chan Xu, Feng Zhao, Qinghong Liu, Xiaojian Qiu, Min Li, Yonghui Yang, Shentong Yu, Huajuan Tong, Lifang Zhang, Bing Chen, Lijuan Qu, Zihua Yu.

  More than 60 monogenic genes mutated in steroid-resistant nephrotic syndrome (SRNS) have been identified. Our previous study found that mutations in nucleoporin 160 kD (NUP160) are implicated in SRNS. The NUP160 gene encodes a component of the nuclear pore complex. Recently, two siblings with homozygous NUP160 mutations presented with SRNS and a nervous system disorder. However, replication of nephrotic syndrome (NS)-associated phenotypes in a mammalian model following loss of Nup160 is needed to prove that NUP160 mutations cause SRNS. Here, we generated a podocyte-specific Nup160 knockout (Nup160podKO) mouse model using CRISPR/Cas9 and Cre/loxP technologies. We investigated NS-associated phenotypes in these Nup160podKO mice. We verified efficient abrogation of Nup160 in Nup160podKO mice at both the DNA and protein levels. We showed that Nup160podKO mice develop typical signs of NS. Nup160podKO mice exhibited progression of proteinuria to average albumin/creatinine ratio (ACR) levels of 15.06 ± 2.71 mg/mg at 26 weeks, and had lower serum albumin levels of 13.13 ± 1.34 g/l at 30 weeks. Littermate control mice had urinary ACR mean values of 0.03 mg/mg and serum albumin values of 22.89 ± 0.34 g/l at the corresponding ages. Further, Nup160podKO mice exhibited glomerulosclerosis compared with littermate control mice. Podocyte-specific Nup160 knockout in mice led to NS and glomerulosclerosis. Thus, our findings strongly support that mutations in NUP160 cause SRNS. The newly generated Nup160podKO mice are a reliable mammalian model for future study of the pathogenesis of NUP160-associated SRNS.

Keywords:  NUP160; knockout; mice; nephrotic syndrome; podocyte

DOI:  https://doi.org/10.1093/hmg/ddad211